10.1016@j.jinorgbio.2018.02.012 (Scopus)

Accepted Manuscript
Sterically modulated silver(I) complexes of coumarin substituted

benzimidazol–2–ylidenes: Synthesis, crystal structures and
evaluation of their antimicrobial and antilung cancer potentials
Gautam Achar, C.R. Shahini, Siddappa A. Patil, Jan Grzegorz

Małecki, Szu-Hua Pan, Albert Lan, Xuan-Ren Chen, Srinivasa
Budagumpi
PII: S0162-0134(17)30615-3
DOI: doi:10.1016/j.jinorgbio.2018.02.012
Reference: JIB 10439
To appear in: Journal of Inorganic Biochemistry
Received date: 29 August 2017
Revised date: 17 February 2018
Accepted date: 18 February 2018
Please cite this article as: Gautam Achar, C.R. Shahini, Siddappa A. Patil, Jan Grzegorz
Małecki, Szu-Hua Pan, Albert Lan, Xuan-Ren Chen, Srinivasa Budagumpi , Sterically
modulated silver(I) complexes of coumarin substituted benzimidazol–2–ylidenes:
Synthesis, crystal structures and evaluation of their antimicrobial and antilung cancer
potentials. The address for the corresponding author was captured as affiliation for all
authors. Please check if appropriate. Jib(2017), doi:10.1016/j.jinorgbio.2018.02.012
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ACCEPTED MANUSCRIPT
Sterically modulated silver(I) complexes of coumarin substituted benzimidazol–

2–ylidenes: Synthesis, crystal structures and evaluation of their antimicrobial
and antilung cancer potentials
Gautam Achara, Shahini C. R.a, Siddappa A. Patila, Jan Grzegorz Małeckib, Szu–Hua Panc,
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Albert Lanc, Xuan–Ren Chenc, Srinivasa Budagumpi*a
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a
Centre for Nano and Material Sciences, Jain University, Jain Global Campus, Kanakapura,
Ramanagaram, Bangalore 562112, India
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b
Department of Crystallography, Institute of Chemistry, University of Silesia, 9th Szkolna
St., 40–006 Katowice, Poland
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c
Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National
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Taiwan University, Taipei 100, Taiwan

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* Corresponding author: Dr. Srinivasa Budagumpi, PhD

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Assistant Professor and Group Leader

Centre for Nano and Material Sciences
Jain University, Jain Global Campus
Kanakapura, Ramanagaram,
Bangalore 562112, India
H/P: +91 9008375705
E–mail: b.srinivasa@jainuniversity.ac.in
dr.sinuvb@gmail.com
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Abstract
In this contribution, a series of sterically–encumbered coumarin substituted benzimidazole–

based N–heterocyclic carbene (NHC) precursors (1–12) and their silver(I)–NHC complexes
(13–24) are reported. Molecular structure of NHC precursors 8 and 12 and cationic
complexes 15 and 16 was established by single crystal X–ray diffraction method. The
silver(I) complexes demonstrated various significant intramolecular agostic–like interactions
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operating between the metal center and the hydrogen atoms of the substituents alongside a
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variety of feeble π–π stacking interactions. A distorted linear coordination geometry is
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documented at the silver(I) center with the anti– arrangement of the ligands. Further, the
complexes demonstrated promising antibacterial properties against Gram positive and Gram
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negative bacterial strains, especially complex 18 displayed a minimum inhibitory
concentration (MIC) of 2 and 4 µg/mL against S. aureas and E. coli, and P. aeruginosa,
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respectively. Furthermore, complexes 14, 15, 16 and 18 were found cytotoxic against the
human lung cancer cell lines A549 and H1975 with the IC50 (concentration of the test sample
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required to kill 50% of the cell population) value under 10 μM, while mono–NHC complex
20 displayed a potential drug window with the IC50 of 13.7±2.70 and 14.5±1.20 μM against
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the cancer cell lines H1975 and A549, respectively. Notably, these complexes displayed
relatively lesser cytotoxic behaviour against the normal skin fibroblast cell line, Hs68. All
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the NHC precursors displayed significantly lower biological activities compared with their
respective complexes, indicating the utility of silver(I) ions in antimicrobial and antilung
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cancer applications.
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Keywords: 4–Bromomethylcoumarin; Antilung–cancer activity; E. coli; N–heterocyclic

carbene; silver(I) complex; X–ray diffraction.
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1. Introduction
Owing to their strong sigma donor ability, the N–heterocyclic carbenes (NHCs) can
coordinate with majority of the d–block elements to form stable transition metal–NHC
complexes that have found extensive in modern organic and organometallic chemistry [1].
NHC derivatives started as a mere research curiosity but have been developed into a class of
handy organic ligands that are used as catalysts in a numerous organic transformation
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reactions [2]. In conjunction with their catalytic applications, NHC–metal complexes have
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been investigated for a wide range of applications that are ranging from catalysis [3,4] to
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biology as anticancer and antimicrobial agents [5]. Because of the ease in structural
modification in NHC scaffolds, great structural diversity is achieved by changing the
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substituents appended at the nitrogen atoms of the central azole core. This structural diversity
has been significantly affected the electronic and steric properties of the NHCs, which in turn
accounts for the variation in the stability, lipophilicity and biocompatibility of the resulting
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complexes. Thus, making NHC based ligands potentially a goldmine for the preparation of
metal–based drug–like molecules in biomedical applications.
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Group X metal complexes, especially platinum derivatives have been primarily used as
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chemotherapeutic candidates [6], however, only recently silver(I) complexes gained focus in
the field of bioinorganic research due to their non–toxic nature. More recently, targeted
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silver(I) based antimicrobial and anticancer preparations are continually increasing, which is
further evidenced by the numerous excellent articles that have been published on this topic
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[7]. Nevertheless, interest on silver(I) in particular is attributed to its ability to target

pathogens selectively, while other xenobiotic metals such as cadmium, mercury, gold etc.
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have been found toxic in their ionic form. Although, the exact mode of action of silver(I)–
NHC complexes is under debate, it is mostly believed that the interaction of silver(I) ions
with thiol containing enzyme of infected cells leading to the deactivation of the enzymatic
protein effectively by curbing the essential metabolic processes that eventually leading to the
cell death [8]. In 2004, Youngs and coworkers reported silver(I) complexes of pincer NHC
ligands that displayed a better bacteriostatic potential than silver(I) nitrate even at a much
lower concentration [9]. This success triggered many researchers to develop a library of
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silver(I) complexes bearing both, functionalized and non–functionalized NHCs for

antimicrobial applications, and later for anticancer potentials. Further, easy functionalization
of the azole ring (imidazole, benzimidazole, triazole and etc.) allowed possible, biologically
active moieties to be tailored on the NHC core. This has been utilized to enhance the potency
and efficacy of the molecule by virtue of both the ligand and the silver(I) ion content.
Recently, Haque and coworkers reported [10] a series of cationic silver(I)–NHC complexes
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that have displayed better anticancer potentials than their gold(I) counterparts. While, a
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gold(III) complex displayed superior activity, with IC50 (concentration of the test sample
required to kill 50% of the cell population) values at low–micro and nanomolar
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concentrations with superior selectivity index.
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In this aspect, both organic and metal–organic derivatives of coumarin heterocycle have long
been studied for their wide spectrum of biological properties such as antibacterial, antifungal
[11], antitumor [12] potentials. The combination of sterically varied benzimidazole core with
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a coumarin substituent are expected to illustrate valuable set of azolium salts for the
preparation of silver(I) complexes, and this prediction is on the basis of higher activity of
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fused or multiheterocyclic derivatives compared with their individual heterocyclic

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derivatives. Recently, we have developed a new class of imidazole–based silver(I)

complexes that displayed an outstanding stability in solution upon exposure to light and
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evidenced excellent antimicrobial activity [13]. It seemed therefore of significant importance

to evaluate the biological potentials of such complexes as potential antimicrobial and
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anticancer agents. Herein, we report our findings on silver(I) complexes of sterically

modulated benzimidazole–based NHC ligands as potent antimicrobial and antilung cancer
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agents. Further, to understand the role of the ancillary ligand, two types of silver(I)
complexes, cationic bis–NHC– and neutral mono–NHC silver(I) derivatives, have been
synthesized.
2. Results and discussion
The benzimidazolium salts and corresponding silver(I)–NHC complexes were prepared in

good yields, and are thoroughly characterized by various spectral and analytical methods. All
the compounds were analyzed for CHN microanalyses and the results are in well agreement
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with the structures established using spectral and single crystal X–ray diffraction methods.
Interestingly, the melting point of salts found decreasing (215–155 oC) as a function of
increase in the alkyl chain length, and the same observation (decomposition onset) was
observed in the case of silver(I) complexes (165–128 oC). The light sensitive dimethyl
sulphoxide soluble, off–white silver(I) complexes were turned either black or beige upon
exposing to light for nearly five days at room temperature. The reason for such color change
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could be the photochemical instability of silver atom; however, the resulted products of
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decomposition were not identified and predicted to be the corresponding benzimidazolium
salts or N–alkyl benzimidazole derivatives along with a form of silver.
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2.1. Synthesis of benzimidazolium salts and silver(I) complexes
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N–alkylated benzimidazole derivatives were prepared by following published literature
procedures [14] with slight modification. The benzimidazole was treated with a strong base
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such as potassium hydroxide in dimethyl sulphoxide at 100oC followed by the addition of an
appropriate alkyl bromide in stoichiometric ratio resulted in the formation of desired N–alkyl
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benzimidazole. Bromination of ethyl acetoacetate in dry ether produced intermediate

bromide, which was treated with 4–methylphenol in acidic medium to afford 4–
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bromomethyl–6–methylcoumarin in good yield [15]. Further, the addition of a 1,4–dioxane

solution of 4–bromomethyl–6–methylcoumarin to a stirring solution of N–alkyl
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benzimidazole in 1,4–dioxane and refluxing for 24 h afforded desired 1,3–disubstituted

benzimidazolium bromide salts, 1–6, as white precipitate. Treating bromide salts 1–6 with
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potassium hexafluorophosphate in methanol : water (9:1 v/v) mixture at room temperature

for 4 h yielded the targeted 1,3–disubstituted benzimidazolium hexafluorophosphate salts, 7–
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12, via salt metathesis reaction as shown in Scheme 1. Both the types of salts were
synthesized in their pure form and hence were used as synthesized for the subsequent
complexation reactions. Bromide salts were readily soluble in polar organic solvents such as
DMSO, DMF, acetonitrile and methanol, while insoluble in diethyl ether and n–hexane;
however, hexafluorophosphate salts were soluble in DMSO, DMF and acetonitrile, while
insoluble in methanol, ethanol, diethyl ether and n–hexane.
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From a synthetic point of view, numerous protocols have been reported for the preparation of
silver(I) NHC complexes; while a particular method is adopted depending on the target
design. Among others, most commonly used method for argentation of azolium salts includes
the direct activation of C–H module present between nitrogen heteroatoms of azole core
using silver(I) oxide or silver(I) acetate as both, metal source as well as the base. The present
sterically encumbered, bis–NHC coordinated silver(I) complexes were prepared following in
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situ deprotonation of azolium salts. Favored by the presence of a non–coordinating anion,
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hexafluorophosphate, bis−NHC silver(I) complexes 13–18 was accomplished directly by
treatment of the benzimidazolium salts 7–12 with silver(I) oxide in acetonitrile for 24 h at 45
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o
C under the exclusion of light as depicted in Scheme 2. So obtained complexes were further
purified by repeated precipitation with acetonitrile and diethyl ether mixture to afford light
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sensitive, air–stable, off–white complexes in good yields. All the complexes were readily
soluble in DMSO, DMF, CHCl3 and acetonitrile, while insoluble in methanol, diethyl ether,
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n–pentane and n–hexane.
Finally, a synthetic approach for the preparation of neutral, mono–NHC coordinated silver(I)
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complexes employing silver(I) acetate using readily available azolium bromide salts has
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ample literature precedence [16]. Following similar synthetic route, the present neutral,
mono–NHC coordinated silver(I) complexes 19–24 having acetate as an ancillary ligand
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were prepared. Scheme 3 represents the synthetic route followed for the preparation of these
complexes. Benzimidazolium bromide salts 1–6 were treated with silver(I) acetate in 1:2
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stoichiometric ratio in methanol at room temperature for 24 h under dark afforded desired
complexes in good yields after recrystallization from methanol and diethyl ether mixture.
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Solubility profile of these complexes was as similar as their hexafluorophosphate

counterparts.
2.2. NMR spectral analyses
Formation of the desired benzimidazolium hexafluorophosphate salts and their

corresponding mono– and bis–NHC coordinated silver(I) complexes was unambiguously
confirmed by proton and carbon NMR spectroscopy over the range δ 0–12 and 0–200 ppm,
respectively. Sterically encumbered salts 7–12 evidenced a characteristic peak in the range δ
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9.7–10.5 ppm was attributed to the resonance of acidic C2 proton, which is a common
observation with other 1,3–disubstituted benzimidazolium salts [17]. The coumarin and
benzimidazolium backbone aromatic protons appeared in the spectra as a set of aromatic
proton resonances in the range δ 7.3–8.2 ppm with appropriate integration value; however,
one apparent triplet and two singlets were observed at around δ 4.50, 6.15 and 5.95 ppm with
appropriate integration values corresponding to alkyl methylene module attached to nitrogen
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atom, coumarin C4 methylene and coumarin ring C3 protons, respectively. Alongside,
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spectra also evidenced the resonance signals in the range δ 0.85–3.6 ppm for all the aliphatic
chain and coumarin C6 methyl protons with well acceptable coupling constants and
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integration values. Interestingly, resonances of coumarin C4 methylene protons attached to
nitrogen atom of benzimidazole appeared shifted by δ 0.45–0.5 ppm toward downfield
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region compared with their imidazole analogues reported by our group earlier [13]. This
apparent shift was due to the presence of benzimidazole moiety that renders the electron
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deficiency in coumarin C4 methylene protons, while chemical shift of C3 proton of coumarin
remained unaltered in all the cases. Three distinct carbon resonance signals were observed at
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round δ 149, 151 and 160 ppm in the corresponding carbon NMR spectra of
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benzimidazolium salts, which are ascribed to the resonances of coumarin ring carbon atoms
attached to the oxygen heteroatoms [18]. Further, a characteristic resonance peak appeared at
around δ 142 ppm corresponding to the resonance of C2 carbon of benzimidazole indicated
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the successful formation of the desired NHC precursors. Similarly, alkyl chain and coumarin
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C4 methylene carbon resonances appeared in the range δ 13–66 ppm, while the aromatic
carbon resonances corresponding to the benzimidazole core and coumarin substituent were
found in the range δ 113–134 ppm. All the spectral observations are in well agreement with
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the analogous compounds reported elsewhere [19].
Obvious indication of the coordination of NHC precursors with silver(I) via deprotonation is
evidenced by the disappearance of the benzimidazolium acidic C2 proton in the proton NMR
spectra of both bis–NHC and mono–NHC coordinated silver(I) complexes (13–24). This
observation is further supported by the appearance of a significantly deshielded C2 carbon
resonance in the region δ 189–191 ppm in the respective carbon NMR spectra of the
complexes, which is in the typical range for the analogous silver(I)–NHC complexes [20].
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However, a few mono–NHC complexes, 19 and 22–24, did not show C2 carbon resonance,
which is evidenced as a common feature in complexes having sterically bulkier NHC ligands
[21]. In the case of mono–NHC complexes 19–24, two new non–NHC peaks were observed
at around δ 24 and 174 ppm, which are corresponding to the methyl and carbonyl carbon
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resonances of the acetate module. Further, no C–107/109Ag coupling was observed in the
carbon NMR spectra of complexes, which enables the present silver(I) complexes to display
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dynamic behavior in solution due to a highly labile carbene carbon–silver(I) bond. Further,
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the carbon NMR spectra of complexes evidenced the resonance peaks that are corresponding
to alkyl chain and aromatic carbon nuclei, which appeared almost unaltered when compared
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with their respective salts.
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2.3. ATR–IR spectral analysis
The availability of functionalities such as lactonic C=O and benzimidazole C=N triggered
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the way for a comparative infrared spectroscopic study of benzimidazolium salts and
corresponding silver(I) complexes. Both, salts and silver(I) complexes, were analyzed by
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ATR–IR spectroscopy in the working IR range of 600–3600 cm–1. Two characteristic strong
stretching bands were observed in the spectra of salts in the range 1700–1740 and 1590–1605
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cm–1, which are ascribed to the vibrations of coumarin lactonic C=O and benzimidazole C=N
modules, respectively. Upon formation of the desired silver(I) NHC complexes, the former
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IR signal due to the stretching vibrations of lactonic carbonyl module found unaltered in the
band position and intensity, which inferred its non–involvement in the coordination sphere.
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Nevertheless, the negative shift observed in the case of benzimidazole C=N stretching bands
indicated the formation of desired complexes. These observations are in line with the similar
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reports found in the literature [22]. Further, aromatic C–H stretching bands for all the
compounds were observed in the range 2900–3000 cm–1 and no broad band at around 3400
cm–1 corresponding to the absence of co–crystalized water molecule. Interestingly, as the
alkyl chain length is increased (from ethyl to hexyl) in the salts and complexes, a distinct
stretching band corresponding to alkyl C–H stretching vibrations was increasingly
intensified, while a shoulder band appeared in the case of benzyl substituted salt 12 and
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respective complexes 18 and 24. These IR observations are well comparable with the similar
azolium salts and corresponding complexes having long alkyl substituents [23].
2.4. Time–dependent NMR spectroscopy studies
With an intention to gain more insight on the solution behavior of the silver(I) complexes, a
representative complex 21 bearing ligand with n–butyl substituent was analyzed for time–
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dependent proton NMR experiments upon exposing the complex solution to light. The proton
NMR chemical shifts that are diagnostic for the complex 21 enable the assignment of other
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species formed upon exposure to light for nearly 10 days at room temperature. The time–
dependent stacked 1H NMR spectra of complex 21 in d6–DMSO solution recorded from day
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1 to 9 are shown in Figure 1. The proton NMR spectrum recorded on day 1 was found in its
purest form with all the resonance peaks well ascribed to each type of proton of the complex
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21 with acceptable integration values. The spectrum evidenced well resolved resonance
signals for butyl chain and methyl protons of coumarin and acetate substituents in the range δ
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0.68–6.14 ppm; while aromatic proton resonances appeared in the range δ 7.27–7.88 ppm.
The stability of the complex 21 in solution was relatively good for first 5 days as no changes
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were observed between the spectra of first and fifth day experiments, indicating that a
DMSO solution of the complex may be used for biological or catalytic applications within
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the first few days of its synthesis. Since the silver atoms in the complex 21 are readily
available to react with light, a slow degradation was observed from day 6 onwards, which
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was evidenced by the appearance of new set of prominent resonance peaks at around δ 8.18,
4.21 and 3.89 ppm. Eventually, the day 9 spectrum depicted the presence of several
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resonance peaks including those observed on day 6, which were like sibling peaks for all the
resonances of complex 21, and no peak observed at around δ 9.30 ppm illustrating the
possible formation of N–butyl benzimidazole along with unidentified derivative of coumarin
and silver(I). This could be presumably due to aquation of the complex solution upon
exposure to light and moisture, which is further evidenced by the appearance of a resonance
peak increasingly broadened at around δ 3.80 ppm. Also, the color of the complex solution in
the NMR tube changed from colorless to black, which prevented further NMR analyses.
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Further, it is highly difficult to characterize the byproducts formed from the test sample after
9 days of exposure to light.
2.5. Molar conductivity studies
In view of the ionic nature of benzimidazolium salts and bis–NHC complexes, the molar
conductivity study was carried out at a test sample concentration of 10–3 M and the data are
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presented in the Experimental Section. Since acetonitrile is a weaker coordinating solvent
than NHC that possess suitable dielectric constant and low viscosity in quantitative terms,
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hence was used as a particular solvent for this study. The molar conductivity value for
benzimidazolium hexafluorophosphate salts fall in the range 139–167 S cm2 mol–1, which is
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well within the range for non–complex ionic compounds possessing 1:1 electrolytic nature.
This observation is well within the range of molar conductance value (an average of 159 S
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cm2 mol–1) for non–complex compounds reported earlier [24]. Alongside, the molar
conductivity results for bis–NHC coordinated silver(I) complexes was in the range 170–195
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S cm2 mol–1, which is attributed to their strong 1:1 electrolytic nature. Comparison of molar
conductivity results of bis–NHC complexes with their corresponding benzimidazolium salts
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revealed the higher ionic mobilities, which presumably due to the involvement of
electropositive silver atoms that display higher conductivity values. While, literature revealed
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that the complexes that are 1:1 electrolytes displayed the molar conductance in a broad
range, 92–199 S cm2 mol–1, measured in acetonitrile [25]. Finally, mono–NHC coordinated
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silver(I) acetate complexes displayed zero molar conductivity as expected, and hence termed
as neutral complexes having a NHC and an acetate ligand system.
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2.6. Single crystal X–ray diffraction studies
Suitable single crystals for X–ray diffraction analysis were isolated for two of the
benzimidazolium salts (8 and 12) and two of the silver(I) complexes (15 and 16), while these
molecular structures were comparable indeed regardless the difference in their N–alkyl
substituents. In general, these benzimidazole–based molecular structures differed only
slightly from our previously reported imidazole and benzimidazole–based carbene precursors
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and complexes [26]. X–ray quality crystals of benzimidazolium salts were obtained by slow
evaporation of a concentrated acetonitrile solution of salt at room temperature for a week,
while that for complexes a lesser temperature (20oC) over a period of about 15 days. The
crystallographic structural data and refinement parameters for both, benzimidazolium salts
and complexes, are presented in Table 1.
Molecular structure of benzimidazolium salts 8 and 12 are shown in Figures 2 and 3 (top),
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respectively. The diffraction data for both the benzimidazolium salts, 8 and 12, were
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completely solved and properly refined in the monoclinic space group P21/c, having
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occupied with one benzimidazolium molecular cation and one hexafluorophosphate anion in
the respective asymmetric unit. Both the salts are well–ordered molecular structures
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crystalized with no solvent molecules and no disorders with the propyl or benzyl
substituents. The arrangements of benzimidazolium cations and hexafluorophosphate anions
are slightly different with a hydrogen bonding interactions. The benzimidazole and coumarin
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rings are planar and almost perpendicular to each other in the former case, while the
coumarin and phenyl rings are planar and placed on both sides of the benzimidazole ring and
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found nearly perpendicular to each other in the latter. The dihedral angle between the planes of
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coumarin and benzimidazole rings differ by small difference, 113.37o for salt 8, and 114.82o
for salt 12; however, angle between benzimidazole ring and propyl or benzyl substituent was
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around 112o for both the salts. The internal ring angle at C1 of the benzimidazole and the
heterocyclic oxygen atoms are 110.85o, 121.56o and 110.79o, 121.91o for salts 8 and 12,
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respectively; which are almost comparable and are in the acceptable range. Correspondingly,
most of the benzimidazole and coumarin ring angles and bond distances were found
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comparable for both the salts. Important bond distances and bond angles for salts 8 and 12
are tabulated in Supporting Information Tables SI1 and SI2, respectively. Observed bond
angles and distances are in well agreement with the similar (benz)imidazolium salts reported
earlier [27]. A comparison in the extended packing motif for salts 8 (left) and 12 (right)
along the b–axis is shown in Supporting Information (Figure SI1). In the extended crystal
structure, a variety of hydrogen bonding interactions and other short contacts were observed.
In the case of salt 8, intramolecular hydrogen bonding interaction was found between the
hexafluorophosphate anion and propyl chain proton with an interaction bond distance of
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2.951 Å (F6–H19A); in conjunction with this, salt 12 displayed similar interactions between
anion and C2 and C4 methylene hydrogen atoms with the bond distance of 2.427 and 2.547
Å (F1–H1 and F2–H15B), respectively. However, no significant stacking interactions were
found in the crystal packing structure of salt 8; while, the salt 12 displayed a feeble π – π
stacking interactions between coumarin ring systems with a centroid to centroid distance of
3.928 Å, as depicted in Figure 3 (bottom).
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The diffraction data for silver(I) complex 15 of NHC bearing n–butyl and coumarin
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substituents were solved and refined in the triclinic space group P–1. The asymmetric unit of
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the complex 15 is composed of one bis–NHC silver(I) complex cation and one
hexafluorophosphate anion. The molecular structure of complex 15 is depicted in Figure 4
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(top), and pertinent bond distances and bond angles are summarized in Table SI3 (Supporting
Information). Complex cation is a well ordered structure; however, two of the fluoride atoms
of hexafluorophosphate anion, F4 and F6, are disordered over two sets. The
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benzimidazolium salt 9 undergone deprotonation to form NHC ligands and coordinated to a
silver(I) ion in an anti– arrangement, adopting a distorted linear coordination geometry
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around the metal center. This particular arrangement of NHC ligands is due to the bulkier
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substituents on one side of the central azole ring, while relatively less steric substituents on
other nitrogen atom [28]. Two NHC ligands linearly coordinated to the silver(I) ion with a
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bond angle of 178.49(15)o (for C1–Ag–C23); and bond distances are being 2.096(4) and
2.091(4) Å for C1–Ag and C23–Ag, respectively. As expected, the internal ring angle at the
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carbene carbon center is 106.3(3)o for N1–C1–N2, which is decreased significantly by almost
4o compared with analogous benzimidazolium salts 8 and 12. This observation inferred the
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coordination of the carbene carbon atoms with the metal center that is in line with similar
observations found in analogous compounds. Further, the internal ring angle at heterocyclic
oxygen atom of coumarin substituents and other bond distances are differed slightly when
compared with salt structures, indicating the non–involvement of the lactonic carbonyl group
in the coordination sphere. Interestingly, four different types of predominant intramolecular
agostic–like interactions are observed between silver(I) and coumarin C4 methylene and
propyl hydrogen atoms (H8A, H19A, H30A and H41A) with the bond distances in the range
2.921–3.102 Å, which are almost equal to the sum of the van der Waals radii of silver and
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hydrogen atoms (3.15 Å). These bond distances and angles are in line with similar silver(I)
complexes reported elsewhere [29]. In the extended crystal structure, adjacent molecules of
complex 15 are linked by hydrogen bonding interactions through hexafluorophosphate
anions with the interaction distances in the range 2.400–2.663 Å. Apparently, this extended
structure is also defined by a feeble face–to–tail π–π stacking interactions [30] operating
between coumarin ring systems with a centroid to centroid distance of 3.937 Å as shown in
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Figure 4 (bottom).
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X–ray diffraction data for complex 16 of NHC bearing n–pentyl and coumarin substituents
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were solved and refined in the triclinic space group P–1. The asymmetric unit of complex 16
is composed with half of a molecule of silver(I) complex cation and half a molecule of
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hexafluorophosphate anion. The molecular structure of complex 16 is shown in Figure 5
(top), and pertinent bond distances and bond angles are summarized in Table SI4 (Supporting
Information). Few of the hydrogen and two carbon atoms of an n–pentyl substituent and a
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fluorine atom in the hexafluorophosphate anion are disordered over two sets. The
benzimidazolium salt 10 undergone deprotonation to form NHC with n–pentyl and coumarin
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substituents and coordinated to silver(I) ion, forming the distorted linear coordination
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geometry around the metal center; which is in well agreement with complex 15. The bond
angle at silver(I) center is being 180.0o for C1–Ag–C1A, and hence both the coordinated
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NHC units are identical. Bond distance for C1–Ag and internal ring angle at the carbene
carbon atom are being 2.094(4) Å and 105.3(4), respectively, which are in acceptable range
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for analogous benzimidazole–based silver(I) NHC complexes [31]. The planes of coumarin
and benzimidazole rings are nearly perpendicular to each other making a dihedral angle of
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112.33o, which is in line with analogous benzimidazolium salts. Strangely, single type of
intramolecular agostic–like interactions were evidenced between the silver(I) and the
coumarin C4 methylene hydrogen atoms with a interaction bond distance of 2.957 Å, which
is lesser than the sum of the van der Waals radii of the silver and hydrogen atoms. The
supramolecular structure of complex 16 differ in many ways (Supporting Information, Figure
SI2) as compared with complex 15, though they are chemically varied by one methylene
group in the alkyl substitution. Three nonequivalent molecules of complex 16 aggregate to
form two predominant face–to–tail π–π stacking interactions between the coumarin–
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coumarin and benzimidazole–benzimidazole ring systems with the centroid to centroid

distance of 3.714 and 3.617 Å, respectively (Figure 5, bottom). Further, these stacking
interactions are complemented by reciprocated chelating hydrogen bonding interactions
between hexafluorophosphate anion and methylene and n–pentyl chain hydrogen atoms; and
coumarin oxygen and methyl hydrogen atoms with the bond distances in the range 2.444–
2.668 Å. Surprisingly, a strong Ag–F1 interaction is observed (along a–axis) with a bond
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distance of 3.063 Å, which is much shorter than the sum of the van der Waals radii of silver
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and fluorine atoms (3.19 Å). All these interactions contributed to a densely packed extended
structure of silver(I) complex 16 containing no trapped solvent or water molecules. The
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molecular structure of these complexes is in well agreement with that of their imidazole
counterparts published earlier by our group [13].
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2.7. Antibacterial and antilung cancer studies
AN
Complexes of group X and XI with biologically relevant ligands have gained the utmost
importance as metal–based chemotherapeutics in the fight against cancer ever since the
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discovery of cisplatin [32]. Finely controlled ligand lability in drawing desired electronic
tuning in the metal center has led to the formation of group X and XI metal NHC complexes
ED
as beneficial candidates in metal–based drug discovery against bacterial infections and

human cancers [33]. The promising biological applications of these NHC complexes arise
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from the controlled release of active species at infected site over a required period of time
[34]. This observation justifies recent synthetic efforts towards the design of new silver(I)
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complexes bearing numerous functionalized and non–functionalized NHC ligands having

varied steric and electronic properties around the metal center are reported as promising
AC
antibacterial and anticancer agents [35]. Among the silver(I) NHC complexes, ionic bis–
NHC and neutral mono–NHC coordinated complexes with the molecular formulations of the
type [NHC–Ag–NHC]X (X: anion, such as halide or hexafluorophosphate) and [NHC–Ag–
OAc], respectively with finely tuned ligand systems have engrossed most attention; in
particular, we [36] and many others [37] have been interested in the biological applications
of silver(I) complexes having functionalized NHC framework. Recently, we have reported a
series of bis –NHC coordinated silver(I) complexes and their antibacterial potentials against
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Gram positive and Gram negative bacterial strains [26b,27a]. Both, azole and alkyl
substituents are varied to understand the role coumarin substituent, and interesting it is
evident that this biologically active substitution is important to achieve the higher activity in
the case of present complexes.
The antibacterial activities of the benzimidazolium salts and corresponding bis–NHC and
mono–NHC silver(I) complexes have been evaluated against a panel of bacterial strains, such
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as S. aureus, B. subtillus, E. coli and P. aeruginosa and the results are compiled in Table 2.
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Under the typical experimental conditions, all the salts were almost inactive with a minimum
CR
inhibitory concentration (MIC) value of more than 128 µg/mL against all the bacterial strains
tested. In contrast, under same experimental conditions, both the types of complexes
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demonstrated excellent to good antibacterial potentials with the MIC value in the range 2–64
µg/mL. All the complexes exhibited impressively low MIC (2–4 µg/mL) values against
Gram–positive bacterial strain S. aureus, while their activity against another Gram–positive
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bacterial strain, B. subtilis, found moderate with the MIC value being either 64 or 32 µg/mL.
In comparison to AgNO3 (MIC: 64 µg/mL against S. aureus and B. subtilis), these complexes
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were found active for a longer period, which presumably rationalized by a slower release of
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active silver(I) ions from the coumarin functionalized derivatives. However, in comparison
to the standard antibacterial drug, Ampicillin, the activity of the present complexes is lesser
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especially against latter bacterial species. On the other hand, both the types of silver(I)
complexes displayed excellent antibacterial activity against the Gram–negative bacteria P.
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aureginosa with the MIC value in the range 4–16 µg/mL, while against E. coli, the activity
was exceptionally good with the MIC value in the range 2–16 µg/mL. Particularly, bis–NHC
AC
complexes 13–16 demonstrated excellent antibacterial activity against latter strain, whereas
all the mono–NHC coordinated complexes 19–24 evidenced a higher MIC value of 16
µg/mL. Interestingly, a bis–NHC coordinated complex 18 featuring benzyl and
methylcoumarin substituent showed a highly promising antibacterial activity against E. coli
strain with a MIC value of 2 µg/mL.
Subsequently, group X and XI metal NHC complexes have been developed as anticancer
agents based on their chemical and biological properties compared to that of cisplatin. All
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new compounds (salts 7–12, bis–NHC 13–18 and mono–NHC 19–24 complexes) were tested
in a SRB (Sulforhodamine B) colorimetric assay to assess their cytotoxicities against the
non–small cell human lung cancer cell lines, H1975 (ATCC® CRL5908™) and A549
(ATCC® CCL–185™). In addition, they were tested for their cytotoxicity against the human
skin fibroblast cell line, Hs68 (ATCC® CRL1635™), as a normal control in all the
experiments. Results of antilung cancer activities of the salts and the silver(I) complexes are
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summarized in Table 3. The SRB colorimetric assay results for bis–NHC and mono–NHC
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coordinated silver(I) complexes are shown in Figures 6 and 7, respectively (for
benzimidazolium salts, Supporting information, Figure SI3). The benzimidazolium salts
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displayed almost no antilung cancer activity against both the cancer cell lines, H1975 and
A549, in the working concentration range of 0.001–10 µM, while, same trend has also been
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observed against the normal skin fibroblast cell line, Hs68. Interestingly, both the types of
silver(I) complexes demonstrated good to moderate antilung cancer activity against H1975
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and A549 cell lines, whereas, less cytotoxic activity against the normal cell line Hs68. Of the
twelve silver(I) NHC complexes synthesized, bis–NHC coordinated complexes 13–18
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displayed the promising activity against the lung cancer cell line A549 with the IC50 value in
the range 8.3±0.40–10 µM. The complex 14 bearing n–propyl and methylcoumarin
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substituents displayed moderate activity against the lung cancer cell line H1975 with an IC50
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value of 9.8±1.0 µM, while other complexes displayed no activity in the working
concentration range of 0.001–10 µM. Further, it is worth mentioning that the cytotoxicity of
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these bis–NHC coordinated complexes found more than 10 µM against the normal skin
fibroblast cell line Hs68. On the other hand, mono–NHC coordinated silver(I) acetate
AC
complexes, 19–24, displayed IC50 value above 10 µM against all the three cell lines, but the
cell survival curves begin to drop in the range 5–10 µM, and therefore these complexes have
been reevaluated for their antilung cancer activity extending the test samples concentration to
20 µM. All these mono–NHC complexes demonstrated a moderate antilung cancer activity
against cancer cell lines H1975 and A549 with the IC50 values in the range 13.2±1.40–
19.4±2.10 and 12.9±1.60–17.7±2.10 µM, respectively; however, their activity against Hs68
cell line closely falls in the same range of 13.1±1.35–18.3±1.55 µM. Of the six mono–NHC
complexes, compounds 19, 20 and 24 displayed lesser activity against normal cell line when
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compared to their activity against cancer cell lines. Lastly, general morphology assessment of
the cancer cells demonstrated the presence of debris and large empty spaces in the cells
treated with complex, while this was not found in the cases of control cells. Nevertheless, the
morphology of the normal skin fibroblast cell line Hs68 treated with complex found almost
same when compared with their normal cells. Especially, the A549 cells shown change in the
morphology with spindle–like structure for complex 20 treated at a concentration of 16 µM
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after 24 h of incubation as shown in Figure 8. It can be concluded with the present antilung
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cancer data, that the bis–NHC complexes were more active than their mono–NHC
counterparts, however at this stage it is difficult draw the conclusions on their structure
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activity relationships.
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3. Conclusions
In summary, two series of silver(I) complexes bearing sterically–modulated coumarin–

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functionalized benzimidazole–based NHC ligands were synthesized and characterized. 1,3–
Disubstituted benzimidazolium bromide salts were prepared by successive N–alkylation
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method, which were converted into their hexafluorophosphate counterparts by following salt
metathesis technique with potassium hexafluorophosphate. Bis– and mono–NHC
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coordinated complexes were prepared by in situ deprotonation of latter salts using silver(I)
oxide and silver(I) acetate, respectively under the exclusion of light. All the compounds were
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completely characterized employing various spectral and analytical techniques. A time

dependent NMR study was conducted to gather more insights on the essential solution
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stability of the silver(I) complexes. The complex found stable in DMSO solution for five
days upon exposure to light and thereafter, a new set of peaks were observed indicating
AC
decomposition of the complex possibly into 1–alkyl benzimidazole and some transient
species of coumarin and silver(I), which are unidentified. Molar conductivity study evidently
distinguished the ionic and neutral nature of bis– and mono–NHC coordinated silver(I)
complexes, respectively. Single crystal X–ray diffraction studies further emphasized the
structure of benzimidazolium salts and complexes. Interestingly, the silver(I) complex
exhibits different types of predominant intramolecular agostic–like interactions that are
almost equal to the sum of the van der Waals radii. In analogy to the wide spread biological
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applications of silver(I) complexes, the present benzimidazolium salts and their complexes
were investigated as potent antibacterial agents against a panel of bacterial strains and
chemotherapeutics against the human lung cancer cells. In both the studies, all the azolium
salts found inactive, while complexes evidenced significant activities. Bis–NHC complexes
proved to possess excellent antimicrobial properties with the MIC value in the range 4–2
µg/mL against S. aureus and E. coli. In the case of antilung cancer studies, both the types of
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silver(I) complexes demonstrated good to moderate antilung cancer activity against the
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cancer cell lines H1975 and A549, whereas, less cytotoxic against the normal skin fibroblast
cell line Hs68. Therefore, these complexes can be further studied in vivo to get more insights
CR
on their antilung cancer applications.
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4. Experimental section
4.1. General considerations

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All the syntheses experiments including argentation were carried out without taking
precautions to exclude air and moisture, while complexes were prepared under the exclusion
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of light. 1–Alkylbenzimidazole derivatives [14] and 4–bromomethyl–6–methylcoumarin [15]

were prepared according to literature methods with slight modification. All chemicals and
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solvents were of reagent grade obtained from commercial sources and used as received
without further distillation or recrystallization. 1H–benzimidazole, ethyl bromide, n–propyl
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bromide, n–butyl bromide, n–pentyl bromide, n–hexyl bromide, benzyl bromide, potassium
hydroxide, potassium hexafluorophosphate, 4–methylphenol, bromine, ethyl acetoacetate,
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silver(I) oxide, silver(I) acetate, nutrient agar and nutrient broth were purchased from
commercial sources. The human lung cancer cell lines NCI–H1975 (ATCC® CRL5908™),
AC
A549 (ATCC® CCL–185™), as well as the human skin fibroblast Hs68 cell line (ATCC®
CRL1635™), were purchased from the American Type Culture Collection (Manassas, VA).
Cells were grown in RPMI or DMEM medium containing 10% FBS and 2 mM L–glutamine
(all from Invitrogen, Eugene, OR) within 5 passages and absence of mycroplasmas at 37°C
in a humidified atmosphere of 5% CO2–95% air. Thin layer chromatography was carried out
on Merck 1.05554 aluminum sheets precoated with silica gel 60 F254 and the compound
spots were observed with UV light at 254 nm or in an iodine chamber. 1H and 13
C NMR
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spectra of compounds were recorded at 298 K in d6–DMSO solvent either on Bruker 300
MHz or Bruker AVANCE III 400 MHz NMR spectrometer. Chemical shifts (δ) are given in
ppm with solvent peaks as the internal references and the multiplicity of the signals is
indicated as singlet (s), doublet (d), triplet (t), and multiplet (m). The melting points were
assessed using a Stuart Scientific (UK) instrument with an accuracy of ±0.3oC. Elemental
analyses were performed using a Perkin Elmer 2400 Series II CHN/S microanalyzer. Infrared
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spectra were recorded on a Bruker ECO–ATR spectrophotometer in the range 600–4000 cm–
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1
. The molar conductivity measurements were made on a digital ELICO–CM–82
conductivity bridge using 0.001 M aqueous potassium chloride solution for calibration.
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4.2. X–ray crystallography
US
For crystals of samples 8, 15 and 16: The crystals were mounted in turn on a Gemini A Ultra
Oxford Diffraction automatic diffractometer equipped with a CCD detector, and used for data
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collection. X–ray intensity data were collected with graphite monochromated MoK radiation (
= 0.71073 Å) at a temperature of 295(2) K, with  scan mode. Lorentz, polarization and
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empirical absorption correction using spherical harmonics implemented in SCALE3 ABSPACK

scaling algorithm [38] were applied. All the non–hydrogen atoms were refined anisotropically
ED
using full–matrix, least–squares technique. All the hydrogen atoms were found from difference
Fourier synthesis after four cycles of anisotropic refinement, and refined as “riding” on the
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adjacent carbon atom with individual isotropic temperature factor equal 1.2 times the value of
equivalent temperature factor of the parent atom. The Olex2 [39] and SHELXS, SHELXL [40]
CE
programs were used for all the calculations. The geometrical calculations were carried out
using the PLATON program. The molecular graphic designs and packing diagram for
AC
publication were performed using Olex2 and MERCURY software packages.
For crystals of sample 12: The X–ray intensity data were collected at a temperature of
296.15 K on a Bruker Proteum2 CCD diffractometer equipped with an X–ray generator
operating at 45 kV and 10 mA, using CuKα radiation of wavelength 1.54178 Å. The
structure was solved by direct methods and refined by full–matrix least squares method on F2
using SHELXS and SHELXL programs [40], while the geometrical calculations were carried
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out using the PLATON program. The molecular graphic designs and packing diagrams of
salt 12 for publication were performed using OLEX2 and MERCURY software packages.
4.3. Synthesis of benzimidazolium salts
4.3.1. Synthesis of 1–ethyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

benzimidazolium bromide/hexafluorophosphate 1/7
To a solution of 5–(bromomethyl)–6–methyl–coumarin (0.5 g, 2 mmol) in 30 mL of
T
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anhydrous 1,4–dioxane, 1–ethylbenzimidazole (0.29 g, 2 mmol) was added drop wise. The
mixture was stirred for 24 h at 80 oC and the white solid thus obtained was filtered, washed
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with fresh anhydrous 1,4–dioxane and dried in air to obtain the benzimidazolium bromide
salt 1. Further, to a methanolic solution (10 mL) of 1 (0.8 g, 2 mmol), 1.5 equivalent of
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potassium hexafluorophosphate (0.552 g) in 10 mL of water–methanol mixture (9:1 v/v) was
added drop wise under continuous stirring. White solid that separated out was allowed to stir
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for further 2 h after which excess distilled water was added to the reaction mixture to remove
unreacted potassium hexafluorophosphate and filtered under suction to afford salt 7. Yield:
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91% M.P.: 215 oC. 1H NMR (400 MHz, d6–DMSO, 298 K): δ 1.60 (3H, t, J = 7.2 Hz, CH3–
ethyl), 2.45 (3H, s, CH3–coumarin), 4.58 (2H, quart, J = 7.2 Hz, CH2–ethyl), 5.97 (1H, s,
ED
C3H–coumarin), 6.16 (2H, s, 4–CH2–coumarin), 7.43 (1HAr, d, J = 8.4 Hz, CH–

benzimidazole), 7.58 (1HAr, d, J = 8.8 Hz, CH–benzimidazole), 7.78–7. 67 (3HAr, m, CH–
PT
benzimidazole/coumarin), 8.04 (1HAr, d, J = 8.0 Hz, CH–coumarin), 8.19 (1HAr, d, J = 8.0

Hz, CH–coumarin), 9.93 (1H, s C2H–benzimidazole). 13C NMR (100 MHz, d6–DMSO, 298
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K): δ 14.3 (CH3–ethyl), 20.9 (CH3–coumarin), 42.9 (CH2–ethyl), 46.8 (C4–coumarin), 113.0,
114.1, 117.0, 117.1, 124.9, 127.2 (ArC–benzimidazole), 114.5, 127.4, 131.7, 131.8, 134.0,
AC
134.4, 149.1, 151.6, 159.9 (ArC–coumarin), 143.4 (C2–benzimidazole). ATR–IR (in cm–1):
3023, 2983 ν(C–H), 1713 ν(C=O, lactonic), 1614 ν(C=N, benzimidazole), 1047 ν(C–O,
coumarin). Anal. Calcd. for C20H19N2O2PF6: C, 51.7; H, 4.1; N, 6.0. Found: C, 51.4; H, 4.2;
N, 5.7. Molar conductance (in acetonitrile, 10–3 M, 302 K): 167 S cm2 mol–1.
4.3.2. Synthesis of 1–propyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

benzimidazolium hexafluorophosphate 2/8
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This NHC precursor was prepared by a method analogous to the earlier synthesis of salt 7.
Yield: 87%, M.P.: 208 oC. 1H NMR (400 MHz, d6–DMSO, 298 K): δ 0.97 (3H, t, J = 7.4 Hz,
CH3–propyl), 2.00 (2H, quart, J = 7.2 Hz, CH2–propyl), 2.44 (3H, s, CH3–coumarin), 4.51
(2H, m, CH2–propyl), 6.00 (1H, s, C3H–coumarin), 6.14 (2H, s, 4–CH2–coumarin), 7.43
(1HAr, d, J = 8.4 Hz, CH–benzimidazole), 7.56 (1HAr, d, J = 8.8 Hz, CH–benzimidazole),
7.77–7.69 (3HAr, m, CH–benzimidazole/CH–coumarin), 8.06 (1HAr, d, J = 8.0 Hz, CH–
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coumarin), 8.20 (1HAr, d, J = 8.4 Hz, CH–coumarin), 9.83 (1H, s, C2H–benzimidazole).
IP
13
C NMR (100 MHz, d6–DMSO, 298 K): δ 11.1 (CH3–propyl), 20.9 (CH3–coumarin), 22.3
(CH2–propyl), 46.8 (CH2–propyl), 48.9 (4–CH2–coumarin), 113.3, 114.5, 117.0, 117.1,
CR
124.8, 127.3 (ArC–benzimidazole), 114.2, 127.4, 131.7, 131.9, 134.0, 134.4, 148.9, 151.6,
159.9 (ArC–coumarin), 143.6 (C2–benzimidazole). ATR–IR (in cm–1): 3016, 2966 ν(C–H),
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1711 ν(C=O, lactonic), 1614 ν(C=N, benzimidazole), 1044 ν(C–O, coumarin). Anal. Calcd.
for C21H21N2O2PF6: C, 52.7; H, 4.4; N, 5.9. Found: C, 52.5; H, 4.8; N, 5.9. Molar
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conductance (in acetonitrile, 10–3 M, 302 K): 160 S cm2 mol–1.
4.3.3. Synthesis of 1–butyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

M

ED
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CH3–butyl), 1.42–1.36 (2H, m, CH2–butyl), 1.97–1.94 (2H, m, CH2–butyl), 2.44 (3H, s,

CH2–coumarin), 4.20–4.17 (2H, m, CH2–butyl), 6.01 (1H, s, C3H–coumarin), 6.21 (2H, s, 4–
CE
CH2–coumarin), 7.42 (1HAr, d, J = 8.4 Hz, CH–benzimidazole), 7.56 (1HAr, d, J = 8.8 Hz,
CH–benzimidazole), 7.77–7.69 (3HAr, m, CH–benzimidazole/coumarin), 8.06 (1HAr, d, J =
AC
8.0 Hz, CH–coumarin), 8.22 (1HAr, d, J = 8.0 Hz, CH–coumarin), 10.01 (1H, s C2H–
benzimidazole). 13
C NMR (100 MHz, d6–DMSO, 298 K): δ 13.9 (CH3–butyl), 19.6 (CH2–
butyl), 20.9 (CH3–coumarin), 30.7 (CH2–butyl), 46.8 (4–CH2–coumarin), 47.3 (CH2– butyl),
113.3, 114.5, 117.0, 117.1, 124.9, 127.3 (ArC–benzimidazole), 114.2, 127.4, 131.7, 131.9,
134.0, 134.4, 148.9, 151.6, 159.9 (ArC–coumarin), 143.6 (C2–benzimidazole). ATR–IR (in
cm–1): 3025, 2954, 2873 ν(C–H), 1711 ν(C=O, lactonic), 1623 ν(C=N, benzimidazole), 1013
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ν(C–O, coumarin). Anal. Calcd. for C22H23N2O2PF6: C, 53.7; H, 4.7; N, 5.7. Found: C, 53.4;
H, 4.8; N, 5.5. Molar conductance (in acetonitrile, 10–3 M, 302 K): 154 S cm2 mol–1.
4.3.4. Synthesis of 1–pentyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

Yield: 89%, M.P.: 168 oC. 1H NMR (400 MHz, d6–DMSO, 298 K): δ 0.89–0.81 (3H, m,
T
CH3–pentyl), 1.36–1.34 (4H, m, CH2–pentyl), 1.97 (2H, m, CH2–pentyl), 2.44 (3H, s, CH3–
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coumarin), 4.56 (2H, t, J = 8.2 Hz, CH2–pentyl), 5.99 (1H, s, C3H–coumarin), 6.20 (2H, s,
CR
4–CH2–coumarin), 7.43 (1HAr, d, J = 8.8 Hz, CH–benzimidazole), 7.58 (2HAr, s, J = 8.4 Hz,
CH–benzimidazole), 7.77–7.68 (3HAr, m, CH–coumarin/benzimidazole), 8.06 (1HAr, d, J =
US
8.0 Hz, CH–coumarin), 8.20 (1HAr, d, J = 8.4 Hz, CH–coumarin), 9.99 (1H, s C2H–
benzimidazole). 13
CNMR (100 MHz, d6–DMSO, 298 K): δ 14.2 (CH3–pentyl), 20.9 (CH3–
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coumarin), 22.0 (CH2–pentyl), 28.3 (CH2–pentyl), 28.4 (CH2–pentyl), 46.9 (CH2–pentyl),
47.3 (4–CH2–coumarin), 113.3, 114.5, 117.1, 124.9, 127.3 (ArC–benzimidazole), 114.2,
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127.4, 131.7, 131.9, 134.0, 134.4, 148.9, 151.6, 159.9 (ArC–coumarin), 143.6 (C2–
benzimidazole). ATR–IR (in cm–1): 3022, 2929, 2858 ν(C–H), 1716 ν(C=O, lactonic),
ED
1622 ν(C=N, imidazole), 1047 ν(C–O, coumarin). Anal. Calcd. for C23H25N2O2PF6: C, 54.5;
H, 5.0; N, 5.5. Found: C, 54.2; H, 5.4; N, 5.7. Molar conductance (in acetonitrile, 10–3 M,
PT
302 K): 160 S cm2 mol–1.
4.3.5. Synthesis of 1–hexyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

CE

AC
CH3–hexyl), 1.36–1.26 (6H, m, CH2–hexyl), 1.98–1.93 (2H, m, CH2–hexyl), 2.44 (3H, s,
CH3–coumarin), 4.50 (2H, t, J = 9.4 Hz, CH2–hexyl), 5.99 (1H, s, C3H–coumarin), 6.20 (2H,
s, 4–CH2–coumarin), 7.41 (1HAr, d, J = 8.4 Hz, CH–benzimidazole), 7.56 (1HAr, d, J = 8.4
Hz, CH–benzimidazole), 7.77–7.68 (3HAr, m, CH–benzimidazole/coumarin), 8.06 (1HAr, d, J
= 7.6 Hz, CH–coumarin), 8.20 (1HAr, d, J = 7.6 Hz, CH–coumarin), 9.99 (1H, s C2H–
benzimidazole). 13
CNMR (100 MHz, d6–DMSO, 298 K): δ 14.3 (CH3–hexyl), 20.9 (CH3–
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coumarin), 22.3 (CH2–hexyl), 25.9 (CH2–hexyl), 28.7 (CH2–hexyl), 31.1 (CH2–hexyl), 46.9
(CH2–hexyl), 47.5 (4–CH2–coumarin), 113.3, 114.5, 117.0, 117.1, 124.9, 127.3 (ArC–
benzimidazole), 114.2, 127.4, 131.7, 131.9, 134.0, 134.4, 148.9, 151.6, 159.9 (ArC–
coumarin), 143.6 (C2–benzimidazole). ATR–IR (in cm–1): 3024, 2964, 2917 ν(C–H), 1714
ν(C=O, lactonic), 1616 ν(C=N, benzimidazole), 1044 ν(C–O, coumarin). Anal. Calcd. for
C24H27N2O2PF6: C, 55.4; H, 5.2; N, 5.4. Found: C, 55.2; H, 5.2; N, 5.6. Molar conductance
T
(in acetonitrile, 10–3 M, 302 K): 162 S cm2 mol–1.
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4.3.6. Synthesis of 1–benzyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–
CR
The NHC precursor was prepared by a method analogous to the earlier synthesis of salt 7.
US
Yield: 80%, M.P.: 252 oC. 1H NMR (400 MHz, d6–DMSO, 298 K): δ 2.43 (3H, s, CH3–
coumarin), 5.80 (3H, s, benzyl–CH2), 6.03 (1H, s, C3H–coumarin), 6.17 (2H, s, 4–CH2–
AN
coumarin), 7.46–7.38 (4HAr, m, benzyl), 7.56–7.55 (2HAr, m, CH–benzimidazole), 7.72–7.66
(3HAr, m, CH–benzimidazole/coumarin), 8.05 (2HAr, m, CH–coumarin), 9.94 (1H, s, C2H–
M
benzimidazole). 13C NMR (100 MHz, d6–DMSO, 298 K): δ 20.9 (CH3–coumarin), 47.0 (4–
CH2–coumarin), 50.7 (CH2–benzyl), 113.4, 114.6, 117.0, 117.1, 124.8, 129.0 (ArC–
ED
benzimidazole), 114.4, 129.3, 129.5, (ArC–benzyl), 127.4, 127.5, 131.7, 134.0, 134.0, 134.4,
148.9, 151.6, 159.9 (ArC–coumarin), 143.9 (C2–benzimidazole). ATR–IR (in cm–1): 3118,
PT
3024 ν(C–H), 1703 ν(C=O, lactonic), 1616 ν(C=N, benzimidazole), 1045 ν(C–O,
coumarin). Anal. Calcd. for C25H21N2O2PF6: C, 57.0; H, 4.0; N, 5.3. Found: C, 56.8; H, 4.3;
CE
4.4. Synthesis of bis–NHC coordinated silver(I) complexes

AC
4.4.1. Synthesis of bis(1–ethyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

benzimidazol–2–ylidenesilver(I) hexafluorophosphate 13
An acetonitrile (25 mL) solution of NHC precursor 7 (0.46 g, 1 mmol) was treated with
silver(I) oxide (0.23 g, 1 mmol) and the mixture was stirred at 45 oC for 24 h under dark. The
reaction mixture was then filtered through a bed of celite and the filtrate was concentrated to
2 mL under reduced pressure. Further, the addition of excess diethyl ether afforded the white
23
ACCEPTED MANUSCRIPT
precipitate with continuous stirring, which upon recrystallization with acetonitrile/ethyl ether
resulted in an off–white solid 15. Yield: 78%, decomposition onset: 165 oC. 1H NMR (400
MHz, d6–DMSO, 298 K): δ 1.40–1.44 (3H, m, CH3–ethyl), 2.39 (3H, s, CH3–coumarin),
4.57–4.52 (2H, m, CH2–ethyl), 5.35 (1H, s, C3H–coumarin), 6.10 (2H, s, CH2–coumarin),
7.32 (1HAr, d, J = 8.4 Hz, CH–benzimidazole), 7.41 (1HAr, d, J = 7.6 Hz, CH–
benzimidazole), 7.49–7.42 (2HAr, m, CH–benzimidazole), 7.75–7.71 (2HAr, m, CH–
T
coumarin), 7.88 (1HAr, d, J = 8.0 Hz, CH–coumarin). 13C NMR (100 MHz, d6–DMSO, 298
IP
K): δ 15.6 (CH3–ethyl), 20.9 (CH3–coumarin), 44.6 (CH2–ethyl), 48.7 (4–CH2–coumarin),
111.8, 112.7, 116.9, 118.4, 123.5, 124.2 (ArC–benzimidazole), 124.7, 124.9, 133.3, 133.6,
CR
131.8, 134.0, 151.4, 152.3, 159.9 (ArC–coumarin), 190.4 (Ag–C2–benzimidazole). ATR–IR
(in cm–1): 2816 ν(C–H), 1718 ν(C=O, lactonic), 1614 ν(C=N, benzimidazole), 1049 ν(C–O,
US
coumarin). Anal. Calcd.for C40H36N4O4AgPF6: C, 54.0; H, 4.1; N, 6.3. Found: C, 53.7; H,
3.8; N, 6.6. Molar conductance (in acetonitrile, 10–3 M, 302 K): 177 S cm2 mol–1.
AN
4.4.2. Synthesis of bis(1–propyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–
M
This complex was prepared in a manner analogous to that of bis–NHC silver(I) complex 13,
ED
with hexafluorophosphate salt 8 (0.47 g, 1 mmol). Yield: 78%, decomposition onset: 162 oC.
1
H NMR (400 MHz, d6–DMSO, 298 K): δ 0.81 (3H, t, J =9.6 Hz, CH3–propyl,), 1.88 (2H,
PT
m, CH2–propyl), 2.41 (3H, s, CH3–coumarin), 4.51 (2H, t, J = 9.2 Hz, CH2–propyl), 5.35
(1H, s, C3H–coumarin), 6.12 (2H, s, 4–CH2–coumarin), 7.34 (1HAr, d, J = 8.0 Hz, CH–
CE

AC
K): δ 11.1 (CH3–propyl), 20.5 (CH3–coumarin), 21.9 (CH2–propyl), 46.1 (CH2–propyl), 48.3
(4–CH2–coumarin), 111.8, 114.5, 116.5, 116.9, 124.3, 126.1 (ArC–benzimidazole), 112.1,
126.9, 130.9, 131.3, 133.4, 134.1, 148.4, 151.3, 159.3 (ArC–coumarin), 190.1 (Ag–C2–
benzimidazole). ATR–IR (in cm–1): 2769 ν(C–H), 1718 ν(C=O, lactonic), 1568 ν(C=N,
benzimidazole), 1094 ν(C–O, coumarin). Anal. Calcd.for C42H40N4O4AgPF6: C, 55.0; H, 4.4;
N, 6.1. Found: C, 54.7; H, 4.7; N, 6.4. Molar conductance (in acetonitrile, 10–3 M, 302 K):
180 S cm2 mol–1.
24
ACCEPTED MANUSCRIPT
4.4.3. Synthesis of bis(1–butyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

with hexafluorophosphate salt 9 (0.49 g, 1 mmol). Yield: 91%, decomposition onset: 160 oC.
1
H NMR (400 MHz, d6–DMSO, 298 K): δ 0.88 (3H, m, J = 8.4 Hz, CH3–butyl), 1.40–1.34
(2H, m, CH2–butyl), 1.95–1.91 (2H, m, CH2–butyl), 2.39 (3H, s, CH3–coumarin), 4.18–4.15
T
(2H, m, CH2–butyl), 5.34 (1H, s, C3H–coumarin), 6.09 (2H, s, 4–CH2–coumarin), 7.35
IP
(1HAr, d, J = 8.2 Hz, CH–benzimidazole), 7.54–7.41 (3HAr, m, CH–benzimidazole), 7.74–
CR
7.72 (2HAr, m, CH–coumarin), 7.91 (1HAr, d, J = 8.0 Hz, CH–coumarin). 13
C NMR (100
MHz, d6–DMSO, 298 K): δ 13.8 (CH3–butyl), 19.5 (CH2–butyl), 20.8 (CH3–coumarin), 30.7
(CH2–butyl), 46.7 (CH2–butyl), 47.3 (4–CH2–coumarin), 111.3, 112.4, 115.0, 122.9, 124.3
US
(ArC–benzimidazole), 112.2, 115.1, 123.4, 129.7, 130.9, 133.0, 133.4, 148.94, 151.6, 159.7
(ArC–coumarin), 191.6 (Ag–C2–benzimidazole). ATR–IR (in cm–1): 2936 ν(C–H), 1719
AN
C44H44N4O4AgPF6: C, 55.9; H, 4.7; N, 5.9. Found: C, 55.7; H, 4.4; N, 6.2. Molar
M

ED
4.4.4. Synthesis of bis(1–pentyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

benzimidazol–2–ylidene silver(I) hexafluorophosphate 16
PT
with hexafluorophosphate salt 10 (0.50 g, 1 mmol). Yield: 74 %, decomposition onset: 155
CE
C. 1H NMR (400 MHz, d6–DMSO, 298 K): δ 0.732–0.68 (3H, m, CH3–pentyl), 1.15–1.12
o
(4H, m, CH2–pentyl), 1.76–1.72 (2H, m, CH2–pentyl), 2.39 (3H, s, CH3–coumarin), 4.49–

AC
4.45 (2H, m, CH2–pentyl), 5.30 (1H, s, C3H–coumarin), 6.12 (2H, s, 4–CH2–coumarin), 7.32
7.51 (2HAr, m, CH–coumarin), 7.88 (1HAr, d, J = 8.0 Hz, CH–coumarin). 13
C NMR (100
MHz, d6–DMSO, 298 K): δ 14.1 (CH3–pentyl), 20.8 (CH3–coumarin), 22.1 (CH2–pentyl),
28.8 (CH2–pentyl), 29.9 (CH2–pentyl), 48.6 (CH2–coumaryl), 49.3 (CH2–pentyl), 111.5,
112.8, 116.9, 117.2, 124.9 (ArC–benzimidazole), 112.6, 124.9, 127.3, 133.7, 133.9, 134.0,
134.2, 151.4, 151.7, 159.7 (ArC–coumarin), 191.1 (Ag–C2–benzimidazole). ATR–IR (in
25
ACCEPTED MANUSCRIPT
cm–1): ~3010, 2928 ν(C–H), 1718 ν(C=O, lactonic), 1613 ν(C=N, benzimidazole), 1046 ν(C–
O, coumarin). Anal. Calcd. for C46H48N4O4AgPF6: C, 56.7; H, 5.0; N, 5.8. Found: C, 57.0; H,
4.4.5. Synthesis of bis(1–hexyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

T
IP
C. 1H NMR (400 MHz, d6–DMSO, 298 K): δ 0.74–0.71 (3H, m, CH3–hexyl), 1.19–1.06
o
CR
(6H, m, CH2–hexyl), 1.75–1.71 (2H, m, CH2–hexyl), 2.39 (3H, s, CH3–coumarin), 4.49–4.45
(2H, m, CH2–hexyl), 5.31 (1H, s, C3H–coumarin), 6.11 (2H, s, 4–CH2–coumarin), 7.32
US
7.71 (2, m, CH–coumarin), 7.88 (1HAr, d, J = 8.0 Hz, CH–coumarin). 13
C NMR (100
AN
MHz, d6–DMSO, 298 K): δ 14.1 (CH3–hexyl), 20.9 (CH3–coumarin), 22.3 (CH2–hexyl), 26.3
(CH2–hexyl), 30.2 (CH2–hexyl), 31.2 (CH2–hexyl), 48.6 (CH2–hexyl), 49.3 (4–CH2–
M
coumarin), 111.4, 112.8, 117.0, 117.2, 124.8, 125.5 (ArC–benzimidazole), 112.6, 124.9,
133.7, 133.9, 134.0, 134.3, 151.5, 151.7, 159.7 (ArC–coumarin), 191.1 (Ag–C2–
ED
benzimidazole). ATR–IR (in cm–1): 2950 ν(C–H), 1722 ν(C=O, lactonic), 1619 ν(C=N,
benzimidazole), 1043 ν(C–O, coumarin). Anal. Calcd for C48H52N4O4AgPF6: C, 57.5; H, 5.2;
PT
N, 5.6. Found: C, 57.1; H, 4.9; N, 5.9. Molar conductance (in acetonitrile, 10–3 M, 302 K):
180 S cm2 mol–1.
CE
4.4.6. Synthesis of bis (1–benzyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

benzimidazol–2–ylidene silver(I) hexafluorophosphate 18
AC
C. 1H NMR (400 MHz, d6–DMSO, 298 K): δ 2.37 (3H, s, CH3–coumarin), 5.78 (3H, s,
o
benzyl–CH2), 5.98 (1H, s, C3H–coumarin), 6.12 (2H, s, 4–CH2–coumarin), 7.29–7.24 (4HAr,

m, benzyl), 7.34–7.31 (2HAr, m, CH–benzimidazole), 7.42–7.38 (3HAr, m, CH–
benzimidazole/coumarin), 7.74 (2HAr, d, J = 8.4 Hz, CH–coumarin). 13
C NMR (100
MHz, d6–DMSO, 298 K): δ 20.9 (CH3–coumarin), 48.9 (CH2–benzyl), 52.8 (4–CH2–
26
ACCEPTED MANUSCRIPT
coumarin), 111.8, 112.7, 113.1, 117.3, 124.9, 128.5 (ArC–benzimidazole), 124.6, 127.7,
128.0 (ArC–benzyl), 117.0, 129.1, 133.8, 133.9, 134.2, 136.6, 151.4, 151.5, 159.7 (ArC–
coumarin), 191.9 (Ag–C2–benzimidazole). ATR–IR (in cm–1): 3079 ν(C–H), 1711 ν(C=O,
lactonic), 1611 ν(C=N, benzimidazole), 1041 ν(C–O, coumarin). Anal. Calcd for
C50H40N4O4AgPF6: C, 59.2; H, 4.0; N, 5.5. Found: C, 59.4; H, 4.2; N, 5.1. Molar
T
4.5. Synthesis of mono–NHC coordinated silver(I) complexes
IP
4.5.1. Synthesis of 1–ethyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–benzimidazol–
CR
2–ylidenesilver(I) acetate 19
To a methanolic solution of benzimidazolium bromide salt 1 (0.319 g, 1 mmol), was added
US
silver(I) acetate (0.33 g, 2 mmol) and the mixture was stirred at room temperature for 24 h
under the exclusion of light. The reaction mixture was then filtered through a bed of ceilte
AN
and the solvent was concentrated to 2 mL under reduced pressure. Further, the addition of
excess diethyl ether with continuous stirring yielded the corresponding neutral, mono–NHC
M
coordinated silver(I) acetate complex 19 as a white solid. Yield: 81 %, decomposition onset:

172 oC. 1H NMR (300 MHz, d6–DMSO, 298 K): δ 0.76–0.76 (3H, m, CH3–ethyl), 1.77 (3H,
ED
s, CH3–acetate), 2.38 (3H, s, CH3–coumarin), 4.49–4.45 (2H, m, CH2–ethyl), 5.40 (1H, s,

C3H–coumarin), 6.14 (2H, s, 4–CH2–coumarin), 7.30 (1HAr, d, J = 8.4 Hz, CH–
PT

CE
K): δ 10.9 (CH3–ethyl), 20.4 (CH3–coumarin), 23.2 (CH3–acetate), 48.2 (CH2–ethyl), 50.2
(4–CH2–coumarin), 111.2, 112.5, 116.5, 116.6, 123.3, 124.4 (ArC–benzimidazole), 112.2,
AC
124.5, 133.3, 134.5, 134.8, 150.9, 151.1, 159.2 (ArC–coumarin), 173.5 (C=O–acetate), Ag–
C2–benzimidazole found absent. ATR–IR (in cm–1): 3056, 2971 ν(C–H), 1718 ν(C=O,
lactonic), 1618 ν(C=N, benzimidazole), 1046 ν(C–O, coumarin). Anal. Calcd. for
C22H21N2O4Ag: C, 54.4; H, 4.4; N, 5.8. Found: C, 54.2; H, 4.8; N, 5.9. Molar conductance
(in acetonitrile, 10–3 M, 302 K): 0 S cm2 mol–1.
4.5.2. Synthesis of 1–propyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–benzimidazol–

27
ACCEPTED MANUSCRIPT
This complex was prepared in a manner analogous to that of mono–NHC silver(I) complex
19, with benzimidazolium bromide salt 2 (0.33 g, 1 mmol). Yield: 82 %, decomposition
onset: 167 oC. 1H NMR (300 MHz, d6–DMSO, 298 K): δ 1.35–1.31 (3H, m, CH3–propyl),
1.64 (3H, s, CH3–acetate), 2.10–2.05 (2H, m, CH2–propyl), 2.38 (3H, s, CH3–coumarin),
4.53–4.50 (2H, m, CH2–propyl), 5.42 (1H, s, C3H–coumarin), 6.14 (2H, s, 4–CH2–
coumarin), 7.29 (1HAr, d, J = 8.4 Hz, CH–benzimidazole), 7.49–7.42 (3HAr, m, CH–
T
benzimidazole), 7.41–7.71 (2HAr, m, CH–coumarin), 7.90 (1HAr, d, J = 7.8 Hz, CH–
IP
coumarin). 13
C NMR (100 MHz, d6–DMSO, 298 K): δ 15.9 (CH3–propyl), 20.3 (CH3–
coumarin), 23.3 (CH3–acetate), 31.1(CH2–propyl), 43.8 (4–CH2–coumarin), 48.3 (CH2–
CR
propyl), 112.5, 113.3, 116.0, 117.1, 124.4, 126.1 (ArC–benzimidazole), 113.5, 126.4, 131.1,
131.9, 133.0, 133.2, 145.4, 151.2,159.3 (ArC–coumarin), 173.4 (C=O–acetate), 189.3 (Ag–
US
C2–benzimidazole). ATR–IR (in cm–1): 3030, 2970 ν(C–H), 1717 ν(C=O, lactonic),
1572 ν(C=N, benzimidazole), 1047 ν(C–O, coumarin). Anal. Calcd. for C23H23N2O4Ag: C,
AN
55.3; H, 4.6; N, 5.6. Found: C, 55.5; H, 4.9; N, 5.8. Molar conductance (in acetonitrile, 10–3
M, 302 K): 0 S cm2 mol–1.
M
4.5.3. Synthesis of 1–butyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–benzimidazol–

ED
This complex was prepared in a manner analogous to that of mono–NHC silver(I) complex
PT
19, with benzimidazolium bromide salt 3 (0.34 g, 1 mmol). Yield: 82 %, decomposition

onset: 163oC. 1H NMR (300 MHz, d6–DMSO, 298 K): δ 0.73–0.65 (3H, m, CH3–butyl),
CE
1.33–1.11 (2H, m, CH2–butyl), 1.67 (3H, s, CH3–acetate), 2.10–2.03 (2H, m, CH2–butyl), 2.

39 (3H, s, CH3–coumarin), 4.52–4.47 (2H, m, CH2–butyl), 5.40 (1H, s, C3H–coumarin), 6.16
AC
(2H, s, 4–CH2–coumarin), 7.31 (1HAr, d, J = 8.4 Hz, CH–benzimidazole), 7.51–7.43 (3HAr,

m, CH–benzimidazole), 7.75–7.73 (3HAr, m, CH–coumarin), 7.91 (1HAr, d, J = 7.8 Hz, CH–
coumarin). 13
C NMR (100 MHz, d6–DMSO, 298 K): δ 14.2 (CH3–butyl), 20.1 (CH3–
coumarin), 21.1 (CH2–butyl), 24.8 (CH3–acetate), 31.3 (CH2–butyl), 49.1 (CH2– butyl),
52.8 (4–CH2–coumarin), 112.8, 113.1, 117.0, 117.3, 124.4, 125.1 (ArC–benzimidazole),
113.6, 126.2, 131.1, 131.4, 134.1, 134.4, 151.1, 151.8, 159.8 (ArC–coumarin), 172.9 (C=O–
acetate), 191.3 (Ag–C2–benzimidazole). ATR–IR (in cm–1): 3052, 2961, 2871 ν(C–H), 1714
28
ACCEPTED MANUSCRIPT
C24H25N2O4Ag:C, 56.1; H, 4.9; N, 5.5. Found: C, 56.5; H, 5.1; N, 5.6. Molar conductance (in
acetonitrile, 10–3 M, 302 K): 0 S cm2 mol–1.
4.5.4. Synthesis of 1–pentyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–benzimidazol–

This complex was prepared in a manner analogous to that of NHC–silver(I) complex 19,
T
with benzimidazolium bromide salt 4 (0.36 g, 1 mmol). Yield: 73 %, decomposition onset:
IP
159 oC. 1H NMR (300 MHz, d6–DMSO, 298 K): δ 0.69–0.65 (3H, m, CH3–pentyl), 1.15–
CR
0.99 (4H, m, CH2–pentyl), 1.68 (3H, s, CH3–acetate), 2.40 (3H, s, CH3–coumarin), 2.50 (2H,
m, CH2–pentyl), 4.50–4.46 (2H, m, CH2–pentyl), 5.36 (1H, s, C3H–coumarin), 6.19 (2H, s,
US
4–CH2–coumarin), 7.32 (1HAr, d, J = 8.4 Hz, CH–benzimidazole), 7.52–7.41 (3HAr, m, CH–
benzimidazole), 7.77–7.74 (2HAr, m, CH–coumarin), 7.91 (1HAr, d, J = 7.8 Hz, CH–
AN
coumarin). 13
C NMR (100 MHz, d6–DMSO, 298 K): δ 13.6 (CH3–pentyl), 20.5 (CH3–
coumarin), 21.7 (CH2–pentyl), 24.9 (CH3–acetate), 28.4 (CH2–pentyl), 28.5 (CH2–pentyl),
M
48.2 (CH2–pentyl), 48.8 (4–CH2–coumaryl), 111.0, 112.3, 116.6, 116.7, 124.4 (ArC–
benzimidazole), 112.5, 124.5, 125.6, 133.3, 133.5, 133.8, 134.4, 151.0, 151.3, 159.2 (ArC–
ED
coumarin), 174.3 (C=O–acetate), Ag–C2–benzimidazole found absent. ATR–IR (in cm–1):

3038, 2852 ν(C–H), 1710 ν(C=O, lactonic), 1569 ν(C=N, benzimidazole), 1047 ν(C–O,
PT
coumarin). Anal. Calcd. for C25H27N2O4Ag: C, 56.9; H, 5.2; N, 5.3. Found: C, 57.3; H, 5.6;
CE
4.5.5. Synthesis of 1–hexyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–benzimidazol–

AC
with benzimidazolium bromide salt 5 (0.37 g, 1 mmol) instead of salt 1. Yield: 69.0%,
decomposition onset: 155 oC. 1H NMR (300 MHz, d6–DMSO, 298 K): δ 0.70–0.68 (3H, m,
CH3–hexyl), 1.14–0.98 (6H, m, CH2–hexyl), 1.68 (3H, s, CH3–acetate), 1.74–1.70 (2H, m,
CH2–hexyl), 2.40 (3H, s, CH3–coumarin), 4.51–4.47 (2H, m, CH2–hexyl), 5.37 (1H, s, C3H–
coumarin), 6.19 (2H, s, 4–CH2–coumarin), 7.32 (1HAr, d, J = 8.4 Hz, CH–benzimidazole),
7.52–7.41 (3HAr, m, CH–benzimidazole), 7.77–7.73 (2, m, CH–coumarin), 7.91 (1HAr, d, J =
29
ACCEPTED MANUSCRIPT
7.8 Hz, CH–coumarin). 13

C NMR (100 MHz, d6–DMSO, 298 K): δ 16.1 (CH3–hexyl), 20.8
(CH3–coumarin), 22.4 (CH2–hexyl), 24.9 (CH3–acetate), 25.9 (CH2–hexyl), 30.2 (CH2–
hexyl), 31.0 (CH2–hexyl), 45.6 (CH2–hexyl), 55.8 (4–CH2–coumarin), 111.3, 112.5, 116.7,
117.0, 124.7 (ArC–benzimidazole), 111.5, 125.6, 133.2, 133.8, 134.2, 134.7, 151.4, 151.7,
159.5 (ArC–coumarin), 174.4 (C=O–acetate), Ag–C2–benzimidazole found absent. ATR–IR
(in cm–1): 3056, 2927 ν(C–H), 1717 ν(C=O, lactonic), 1573 ν(C=N, imidazole), 1048 ν(C–O,
T
coumarin). Anal. Calcd. for C26H29N2O4Ag: C, 57.7; H, 5.4; N, 5.2. Found: C, 57.8; H, 5.6;
IP
CR
4.5.6. Synthesis of 1–benzyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–benzimidazol–
US
with benzimidazolium bromide salt 6 (0.414 g, 1 mmol). Yield: 72 %, decomposition onset:
AN
182 oC. 1H NMR (300 MHz, d6–DMSO, 298 K): δ 1.60 (3H, s, CH3–acetate), 2.34 (3H, s,
CH3–coumarin), 5.44 (1H, s, C3H–coumarin), 5.76 (3H, s, benzyl–CH2), 6.18 (2H, s, 4–
M
CH2–coumarin), 7.21–7.16 (4HAr, m, benzyl), 7.33–7.31 (2HAr, m, CH–benzimidazole),

7.46–7.38 (3HAr, m, CH–benzimidazole/coumarin), 7.71–7.69 (2HAr, m, CH–coumarin). 13C
ED
NMR (100 MHz, d6–DMSO, 298 K): δ 20.3 (CH3–coumarin), 24.9 (CH3–acetate), 48.5 (4–
CH2–coumarin), 51.9 (CH2–benzyl), 111.4, 112.3, 116.5, 124.3 (ArC–benzimidazole), 112.6,
PT
124.5, 127.3 (ArC–benzyl), 116.6, 127.9, 128.6, 133.2, 134.3, 136.1, 150.8, 151.0, 159.2
(ArC–coumarin), 174.1 (C=O–acetate), Ag–C2–benzimidazole found absent. ATR–IR (in
CE
cm–1): 3167, 3068 ν(C–H), 1709 ν(C=O, lactonic), 1617 ν(C=N, benzimidazole), 1019 ν(C–
O, coumarin). Anal. Calcd. for C27H23N2O4Ag:C, 59.2; H, 4.2; N, 5.1. Found: C, 59.6; H,
AC
4.6. Antimicrobial analyses
The antibacterial potentials of the prepared benzimidazolium hexafluorophosphate salts (7–

12) and their bis– and mono–NHC silver(I) complexes (13–24) were evaluated using the
broth dilution method against a panel of Gram positive, S. aureus and B. subtilis, and Gram
negative E. coli and P. aeruginosa, bacterial strains at different concentrations. Minimum
inhibitory concentration (MIC) of compounds 7–24 was determined against both the types of
30
ACCEPTED MANUSCRIPT
bacteria in three individual experiments and any two concurrent values are presented. The
MIC of the benzimidazolium salts and silver(I) complexes was determined by serially
diluting the test compounds 7–24 to obtain a concentration range of 1–256 µg/mL which was
further diluted by adding 1 mL bacterial suspension to obtain a final concentration range
from 0.5–128 µg/mL and a final desired inoculum of 5×105 CFU mL–1. The control tubes
contained 10% DMSO mixture in nutrient broth along with the bacterial suspension. All the
T
test tubes were incubated at 37oC for 16 h. MIC was read as the lowest concentration of the
IP
test compound that inhibited visible bacterial growth.
CR
4.7. Anticancer analyses
4.7.1. Cell lines and culture conditions
US
The antilung cancer potentials of the prepared benzimidazolium hexafluorophosphate salts
(7–12) and their bis– and mono–NHC silver(I) complexes (13–24) were evaluated against the
AN
human lung cancer and human skin fibroblast cell lines. The human lung cancer cell lines,
NCI–H1975 (ATCC® CRL5908™) and A549 (ATCC® CCL–185™), as well as the human
M
skin fibroblast Hs68 cell line (ATCC® CRL1635™), were purchased from the American
Type Culture Collection (Manassas, VA). The human skin fibroblast cell line, Hs68 was used
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as the normal control. Cells were grown in RPMI or DMEM medium containing 10% FBS
and 2 mM L–glutamine (all from Invitrogen, Eugene, OR) within 5 passages and absence of
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mycoplasma at 37 °C in a humidified atmosphere of 5 % CO2–95 % air. All the anticancer

experiments were ran simultaneously against the same cell lines considering a purine type
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derivative (GRC0321) that acts as a microtubule targeting agent as a positive control (IC50:
0.30±0.03 µM against H1975) [42] and DMSO as a negative control.
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4.7.2. SRB colorimetric assay for cytotoxicity screening
For SRB assay [41], healthy human lung cancer and skin fibroblast cells were used. Prior to
the experiment, 103 cells were cultured in 96–well culture plates for 24 h. The culture
medium was replaced with fresh medium containing different dosages of test compounds (10
µM, 5 µM, 1 µM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM and DMSO) for 72 h. In the case
of mono–NHC coordinated silver(I) acetate complexes a concentration of 20 µM was tried.
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Cells were fixed for 30 min at room temperature with 10% trichloroacetic acid (TCA) and
were stained for 30 min with 0.4 % (wt/vol) sulforhodamine B (SRB) dissolved in 1 % acetic
acid after washing five times with water. Unbound dye was removed by three washes with 1
% acetic acid, and protein–bound dye was extracted with 10 mM unbuffered Tris base,
[tris(hydroxymethyl)aminomethane], for determination of optical density in a computer
interfaced, 96–well microtiter plate reader. OD was measured at 570 nm and growth curves
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were plotted using GraphPad software (San Diego, CA); and the IC50 of each test compound
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was obtained using linear interpolation. Cell survival was calculated using the equation (1).
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IC50 = [(Ti–C)/C] x 100 …..(Eq. 1)
Where, C: control growth; Ti: test growth in the presence of test compound at 8
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concentration levels.
Acknowledgments
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S.B. thanks Science and Engineering Research Board–Department of Science and
Technology, New Delhi, India for the financial support through the Young Scientist Start Up
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Research Grant, YSS/2014/000032. The authors are grateful to the Institution of Excellence,
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Vijnana Bhavana, University of Mysore, India and Jain University, India, for providing the
single–crystal X–ray diffractometer (for benzimidazolium salt 12) and spectral facilities,
respectively. S. –H. P. also thanks for the grants supported from Academia Sinica and
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Ministry of Science and Technology (Taiwan) (MOST 104–0210–01–09–02 and 105–2628–

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B–002–007–MY3).
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Scheme 1: Synthesis of sterically–tuned coumarin–tethered benzimidazole–based NHC

precursors 1–12.
Scheme 2: Synthesis of cationic, bis–NHC coordinated silver(I) hexafluorophosphate

complexes, 13–18.
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Scheme 3: Synthesis of neutral, mono–NHC coordinated silver(I) acetate complexes 19–24.
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Figure 1. Time–dependency of the 1H NMR spectra of a d6–DMSO solution of silver(I)
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complex 21. The NMR spectra of complex 21 were recorded every day upon exposure to
sunlight (D1: day 1, D2: day 2, D3: day 3, D4: day 4, D5: day 5, D6: day 6, D7: day 7, D8:
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day 8 and D9: day 9).
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Figure 2. Molecular structure of NHC precursor 8. Important bond distances (Å): N1–C1
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1.326(3); N2–C1 1.322(3); O1–C11 1.371(3); O1–C12 1.375(3); O2–C11 1.204(3).

Important bond angles (o): N1–C1–N2 110.8(2); C11–O1–C12 121.56(17).
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Figure 3. Molecular structure of NHC precursor 12 (top) and π – π stacking interactions

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operating between coumarin ring systems (centroid to centroid distance of 3.928 Å) (bottom,
hydrogen atoms and hexafluorophosphate anions are omitted for clarity). Important bond
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distances (Å): N1–C1 1.319(4); N2–C1 1.329(4); O1–C18 1.370(4); O1–C19 1.389(3); O2–
C18 1.215(4). Important bond angles (o): N1–C1–N2 108.5(2); C18–O1–C19 121.9(2).
Figure 4. Molecular structure of complex 15 (top) and π – π stacking interactions operating

between coumarin ring systems (centroid to centroid distance of 3.937 Å) (bottom).
Hydrogen atoms and hexafluorophosphate anions have been omitted for clarity. Important
bond distances (Å): Ag1–C1 2.096(4); N1–C1 1.355(5); N2–C1 1.347(5); O1–C11 1.374(5);
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O2–C11 1.211(5). Important bond angles (o): C1–Ag1–C23 178.49(15); N1–C1–N2

106.3(3); C11–O1–C12 121.8(3).
Figure 5. Molecular structure of complex 16 (top) and π – π stacking interactions operating

between coumarin ring (centroid to centroid distance of 3.714 Å) and benzimidazole ring
systems (centroid to centroid distance of 3.617 Å) (bottom). Hydrogen atoms and
hexafluorophosphate counterion have been omitted for clarity. Important bond distances (Å):
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Ag1–C1 2.094(4); N1–C1 1.358(6); N2–C1 1.348(5); O1–C11 1.373(7). Important bond
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angles (o): C1–Ag1–C1A 180.0; C11–O1–C12 121.8(4).
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Figure 6. Cell survival curves of the bis–NHC coordinated silver(I) complexes 13–18 against
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Hs68, H1975 and A549 cell lines. AN
Figure 7. Cell survival curves of the mono–NHC coordinated silver(I) complexes 19–24
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against Hs68, H1975 and A549 cell lines.

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Figure 8. Images showing cell morphology of Hs68, NCI–H1975 and A549 cell lines treated
with silver(I) complex 20 (16 µM) for 24 h of incubation.
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Table 1. Crystallographic data and the structure refinement details for the NHC precursors 8
and 12 and silver(I)–NHC complexes 15 and 16.
8 12 15 16
C21H21N2O2PF C44H44AgN4O4P C46H46AgN4O4P
Formula C25H21N2O2PF6
6 F6 F6
Formula weight 478.37 1052.82 945.67 971.71
Crystal system Monoclinic Monoclinic Triclinic Triclinic
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Space group P21/c P21/c P-1 P-1
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Unit cell dimensions
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a (Å) 10.0322(5) 9.9126(4) 11.3263(5) 7.8575(6)
b (Å) 14.5418(7) 27.3572(12) 13.0821(6) 11.4817(10)
c (Å) 14.9334(9) 9.6945(4) 15.0130(7) 13.9198(13)
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α (°) 90 90.00 93.362(4) 105.625(8)
β (°) 95.051(5) 118.017(2) 108.304(4) 105.174(7)
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γ (°) 90 90.00 91.738(4) 105.992(7)
V (Å3) 2170.1(2) 2320.87(17) 2105.53(17) 1083.35(19)
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Z 4 2 2 1
Density(calcd)
1.464 1.507 1.501 1.489
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(g/cm3)
Abs. coeff. (mm-1) 0.199 1.738 0.591 0.576
F(000) 984 1080 968 498
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0.16x0.12x0.0 0.28x0.26x0.2
Crystal size (mm) 0.32x0.19x0.04 0.39x0.18x0.06
5 2
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Temperature (K) 295(2) 296(2) 295(2) 295(2)

Radiation (Å) 0.71073 1.54178 0.71073 0.71073
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θ Min, Max (°) 3.465-29.401 3.23-64.53 3.8940-26.2180 3.6880-24.1280

-10:13, -15:19, -11:11, -31:30, -13:14, -17:17, - -9:10, -15:15, -
Data set
-19:14 -11:10 20:19 18:19
Tot., Uniq. Data 10349 12556 10022 10527
R (int) 0.0515 0.0516 0.0610 0.0775
Nref, Npar 5135, 291 3716, 326 6388, 565 5050, 315
0.0515, 0.0516, 0.0610, 0.1407, 0.0776, 0.1769,
R, wR2, S
0.1165, 1.022 0.1357, 1.125 1.017 1.098
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Table 2. Antimicrobial efficacies of bis– and mono–NHC silver(I) complexes 13-24 in terms
of MIC (in µg/mL) against a panel of bacterial strains.
Compound S. aureus B. subtillus E. coli P. aeruginosa

13 4 128 4 8
14 4 64 4 8
15 4 64 4 8
4 64 4 8
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16
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17 4 32 8 8
18 2 32 2 4
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19 4 32 16 8
20 4 32 16 8
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21 4 32 16 8
22 4 32 16 8
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23 4 32 16 16
24 4 32 16 8
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Ampicillin ≤0.5 ≤0.5 4 ----

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Table 3. Antilung cancer efficacies of NHC precursors and bis– and mono–NHC silver(I)
complexes 7-24 in terms of IC50 (in μM) values against Hs68, H1975 and A549 cell linesa.
Compound Hs68 H1975 A549

13 9.4±0.30 >10 8.4±0.80
14 7.7±1.65 9.8±1.0 8.3±0.40
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15 >10 >10 9.0±0.50
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16 >10 >10 8.6±0.70
17 >10 >10 >10
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18 >10 >10 8.7±0.65
19 18.3±1.55 13.8±1.80 17.7±2.10

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20 17.9±1.90 13.7±2.70 14.5±1.20
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21 14.4±1.80 15.2±1.20 13.3±1.80

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22 14.0±1.90 19.4±2.10 14.1±1.30

23 14.1±1.05 14.7±2.10 17.7±1.00
24 13.1±1.35 13.2±1.40 12.9±1.60
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a
: Azolium salts 7–12 displayed no antilung cancer activity against any of the cell lines tested
at a concentration of 10 μM.
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Highlights:
 A series of coumarin substituted benzimidazolium salts were reported

 Two series of bis- and mono-NHC coordinated Ag complexes were prepared
 All three series of compounds were fully characterized
 Some of the Ag complexes displayed promising antimicrobial activities
 Bis-NHC Ag complexes evidenced promising antilung cancer activities
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Graphics Abstract
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

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Sterically modulated silver(I) complexes of coumarin substituted

Gautam Achar, C.R. Shahini, Siddappa A. Patil, Jan Grzegorz

Sterically modulated silver(I) complexes of coumarin substituted benzimidazol–

Taiwan University, Taipei 100, Taiwan

* Corresponding author: Dr. Srinivasa Budagumpi, PhD

Assistant Professor and Group Leader

In this contribution, a series of sterically–encumbered coumarin substituted benzimidazole–

Keywords: 4–Bromomethylcoumarin; Antilung–cancer activity; E. coli; N–heterocyclic

[7]. Nevertheless, interest on silver(I) in particular is attributed to its ability to target

silver(I) complexes bearing both, functionalized and non–functionalized NHCs for

fused or multiheterocyclic derivatives compared with their individual heterocyclic

derivatives. Recently, we have developed a new class of imidazole–based silver(I)

evidenced excellent antimicrobial activity [13]. It seemed therefore of significant importance

anticancer agents. Herein, we report our ﬁndings on silver(I) complexes of sterically

2. Results and discussion

The benzimidazolium salts and corresponding silver(I)–NHC complexes were prepared in

benzimidazole. Bromination of ethyl acetoacetate in dry ether produced intermediate

bromomethyl–6–methylcoumarin in good yield [15]. Further, the addition of a 1,4–dioxane

benzimidazole in 1,4–dioxane and refluxing for 24 h afforded desired 1,3–disubstituted

potassium hexafluorophosphate in methanol : water (9:1 v/v) mixture at room temperature

Solubility profile of these complexes was as similar as their hexafluorophosphate

2.2. NMR spectral analyses

Formation of the desired benzimidazolium hexafluorophosphate salts and their

the analogous compounds reported elsewhere [19].

2.4. Time–dependent NMR spectroscopy studies

2.5. Molar conductivity studies

2.6. Single crystal X–ray diffraction studies

coumarin and benzimidazole–benzimidazole ring systems with the centroid to centroid

as beneficial candidates in metal–based drug discovery against bacterial infections and

complexes bearing numerous functionalized and non–functionalized NHC ligands having

In summary, two series of silver(I) complexes bearing sterically–modulated coumarin–

completely characterized employing various spectral and analytical techniques. A time

4.1. General considerations

of light. 1–Alkylbenzimidazole derivatives [14] and 4–bromomethyl–6–methylcoumarin [15]

empirical absorption correction using spherical harmonics implemented in SCALE3 ABSPACK

publication were performed using Olex2 and MERCURY software packages.

4.3. Synthesis of benzimidazolium salts

4.3.1. Synthesis of 1–ethyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

To a solution of 5–(bromomethyl)–6–methyl–coumarin (0.5 g, 2 mmol) in 30 mL of

C3H–coumarin), 6.16 (2H, s, 4–CH2–coumarin), 7.43 (1HAr, d, J = 8.4 Hz, CH–

benzimidazole/coumarin), 8.04 (1HAr, d, J = 8.0 Hz, CH–coumarin), 8.19 (1HAr, d, J = 8.0

4.3.2. Synthesis of 1–propyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

4.3.3. Synthesis of 1–butyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

benzimidazolium hexafluorophosphate 3/9

CH3–butyl), 1.42–1.36 (2H, m, CH2–butyl), 1.97–1.94 (2H, m, CH2–butyl), 2.44 (3H, s,

4.3.4. Synthesis of 1–pentyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

302 K): 160 S cm2 mol–1.

4.3.5. Synthesis of 1–hexyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

benzimidazolium hexafluorophosphate 5/11

4.4. Synthesis of bis–NHC coordinated silver(I) complexes

4.4.1. Synthesis of bis(1–ethyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

benzimidazole), 7.53–7.43 (3HAr, m, CH–benzimidazole), 7.76–7.74 (2HAr, m, CH–

4.4.3. Synthesis of bis(1–butyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

conductance (in acetonitrile, 10–3 M, 302 K): 195 S cm2 mol–1.

4.4.4. Synthesis of bis(1–pentyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

(4H, m, CH2–pentyl), 1.76–1.72 (2H, m, CH2–pentyl), 2.39 (3H, s, CH3–coumarin), 4.49–

4.4.5. Synthesis of bis(1–hexyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

4.4.6. Synthesis of bis (1–benzyl–3–((6–methyl–2–oxo–2H–chromen–4–yl)methyl)–

benzyl–CH2), 5.98 (1H, s, C3H–coumarin), 6.12 (2H, s, 4–CH2–coumarin), 7.29–7.24 (4HAr,

To a methanolic solution of benzimidazolium bromide salt 1 (0.319 g, 1 mmol), was added

coordinated silver(I) acetate complex 19 as a white solid. Yield: 81 %, decomposition onset:

s, CH3–acetate), 2.38 (3H, s, CH3–coumarin), 4.49–4.45 (2H, m, CH2–ethyl), 5.40 (1H, s,